While ethylene is produced from crude petroleum or natural gas, it has long been known that ethylene could also be formed by plants and microorganisms.
Chou et al. reported that .alpha.-ketoglutaric acid(.alpha.-KG) or L-glutamic acid(Glu) is a precursor of ethylene in Penicillium digitatum, a species of fungi (Arch. Biochem. Biophys., 157, 73, 1973), and Goto et al. reported that .alpha.-KG serves as a substrate in the ethylene forming system in cell-free extracts of Pseudomonas syringae which is one of the species of pathogenic bacteria for plants (Plant Cell Physiol., 28, 405, 1987).
Primrose et al. reported that, in the ethylene-forming system in cell-free extracts of Escherichia coli, 2-keto-4-methylthiobutyric acid(KMBA) which is a metabolic intermediate of L-methionine(Met) is a precursor of ethylene biosynthesis (J. Gen. Microbiol.,98,519, 1977). In all these reports, however, the true substrates and biosynthetic pathway of ethylene-forming reaction were not established because materials such as cultured cells of bacteria or their cell free extracts, which are supposed to contain a lot of impurities, were used in the experiments.
To make clear the pathway of the ethylene biosynthesis caused by the bacteria and the ethylene-forming enzyme reaction, the present inventors purified the enzyme catalyzing the ethylene formation to electrophoretically homogeneous state. The results revealed that the ethylene-forming reaction via KMBA is really radical reactions in which active oxygen is concerned (Fukuda et al., FEMS Microbiol. Lett., 60, 107, 1989). We have also investigated the ethylene-forming enzyme of Penicillium digitatum IFO 9372 via a .alpha.-KG and its enzymatic reaction (Fukuda et al., FEMS Microbiol.lett., 59, 1989), and the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 via .alpha.-KG and its characteristics (Nagahama, Fukuda et al., J. Gen. Microbiol., 137, 2228, 1991).
Although the enzyme catalyzing the ethylene biosynthesis by bacteria was identified as well as the characteristics of the enzyme, by the present inventors as described above, the ability of ethylene formation in these ethylene-forming bacteria was not adequately sufficient.
For the purpose of fundamental breeding improvement of the ethylene-forming bacteria through a gene manipulation technique, Pseudomonas syringae pv. phaseolicola PK2 was selected for the object and its DNA sequence encoding the ethylene-forming enzyme was analyzed, thereby completing this invention.